a, 2N chimaeras generated with STAP cells derived from Oct4-gfp C57BL/6 mice (left) and 129/Sv×C57BL/6 F1 mice (right). b, Generation of chimaeric mice from STAP cells by cluster injection. STAP cells used in the experiments above were generated from CD45+ lymphocytes of multiple neonatal spleens (male and female tissues were mixed). *All fetuses were collected at 13.5d.p.c. to 15.5d.p.c. and the contribution rate of STAP cells into each organ was examined by FACS. **The contribution of STAP cells into each chimaera was scored as high (>50% of the coat colour of GFP expression). ***B6GFP: C57BL/6 mouse carrying cag-gfp.c, Production of offspring from STAP cells via germline transmission. Chimaeras generated with 129/Sv×B6GFP STAP cells (obtained from the experiments shown in b) were used for germline transmission study. d, 4N embryos at E9.5 generated with STAP cells derived from F1 GFP mice (B6GFP and DBA/2 or 129/Sv). B6GFP, C57BL/6 mouse carrying cag-gfp.

Present address: Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi 400-8510, Japan.

Teruhiko Wakayama

Contributions

H.O. and Y.S. wrote the manuscript. H.O., T.W. and Y.S. performed experiments, and K.K. assisted with H.O.’s transplantation experiments. H.O., T.W., Y.S., H.N. and C.A.V. designed the project. M.P.V. and M.Y. helped with the design and evaluation of the project.

a, Immunostaining for Ki67 and BrdU. STAP cell clusters (top) and ES cell colonies (bottom) are shown. For BrdU uptake, BrdU was added into each culture medium (10μM) for 12h until fixation. Scale bar, 100μm. b, Transformation assay by soft agar culture. Neither Oct4-GFP+ nor Oct4-GFP-dim cells showed colony formation in soft agar, whereas ES cells and STAP stem cells showed anchorage-independent growth in the same LIF-B27 medium. Scale bar, 100µm. Proliferated cells were lysed and the amount of DNA in each well was estimated by chemical luminescence (graph). n = 3 , average±s.d. c, No substantial change in chromosome number was seen with STAP cells in the CGH array. Genomic DNA derived from CD45+ cells (male) was used as reference DNA. The spikes (for example, those seen in the X chromosome) were nonspecific and also found in the data of these parental CD45+ cells when the manufacturer’s control DNA was used as a reference. d, qPCR analysis for pluripotency markers that highly express in ES cells, but not in EpiSCs. Average±s.d. e, Immunostaining of markers for mouse EpiSC and ES cells. Scale bar, 100 μm. f, g, H3K27me3+ foci in female cells, which are indicative of X-chromosomal inactivation. These foci were not observed in male cells. Scale bar, 10μm. In the case of female STAP cells, ~40% of cells retained H3K27me3+ foci (g). **P<0.001; Tukey’s test. n = 3, average±s.d. Although nuclear staining looked to be higher in STAP cells with H3K27me3+ foci (f), this appeared to be caused by some optical artefacts scattering from the strong focal signal. h, qPCR analysis for the tight junction markers Zo-1 and claudin 7, which were highly expressed in EpiSCs, but not in ES cells or STAP cells. **P<0.01; ns, not significant; Tukey's test; n = 3, average±s.d.

a, 2N chimaeras generated with STAP cells derived from Oct4-gfp C57BL/6 mice (left) and 129/Sv×C57BL/6 F1 mice (right). b, Generation of chimaeric mice from STAP cells by cluster injection. STAP cells used in the experiments above were generated from CD45+ lymphocytes of multiple neonatal spleens (male and female tissues were mixed). *All fetuses were collected at 13.5d.p.c. to 15.5d.p.c. and the contribution rate of STAP cells into each organ was examined by FACS. **The contribution of STAP cells into each chimaera was scored as high (>50% of the coat colour of GFP expression). ***B6GFP: C57BL/6 mouse carrying cag-gfp.c, Production of offspring from STAP cells via germline transmission. Chimaeras generated with 129/Sv×B6GFP STAP cells (obtained from the experiments shown in b) were used for germline transmission study. d, 4N embryos at E9.5 generated with STAP cells derived from F1 GFP mice (B6GFP and DBA/2 or 129/Sv). B6GFP, C57BL/6 mouse carrying cag-gfp.

a, Percentages of Oct4-GFP-expressing cells 7days after stress treatment. Somatic cells were isolated from various tissues and exposed to different stressors. Oct4-GFP expression was analysed by FACS. b, Oct4 and Oct4-GFP expression induced in the reflux oesophagitis mouse model as an in vivo acid exposure model (top, experimental procedure). Oct4, but not Nanog, expression was observed in the oesophageal epithelium exposed to gastric acid (75% of 12 operated mice).

DIC images during day 0 – day 7, overlaid with oct3/4::GFP (green). A strong contrast of DIC (as compared to video 2) was applied to imaging so that lamellipodia-like processes (frequently seen on and after day 4) could be viewed easily.

DIC images during day 0 – day 6, overlaid with oct3/4::GFP (green). The interval of imaging was half (15 min) of that of video 1 (the overall speed of the video is three-times slower than video 1). In this view field where the cell density was relatively low, behaviours of individual cells were more easily seen. In this case, forming clusters were slightly smaller in size.